Crystalline form of riboflavin



July 15, 1952 Filed Jan. 26, 1950 SOLUBlLlTY CHARACTERISTICS OF THE THREE CRYSTAL TYPES OF RIBOFLAVIN TYPE C J. K. DALE 2,603,633

CRYSTALLINE FORM OF RIBOFLAVIN 2 SHEETSSHEET 1 400 @00 aoo Ms /L TAKEN FIGURE Mc IN SOLUTION INVENTOR ATTORNEY July 15, 1952 J.- K. DALE CRYSTALLINE FORM '0? RIBOFLAVIN Filed Jan. 26, 1.950

2 SHEETS-SHEET 2 $.03. m a w n d w M r 29292 22 M2 95 @o no 8 2w Q0: wo no I. No 6 o 52:23 EEC 23;

I NV E NTO R A T T 0 RN E Y I Patented July 15, 1952 NIT D" CRYSTALLINE FORM OF'RIBOFLAVIN' Julian K. Dale, ,Terre 'Haute, Ind., assignor ,to

Commercial Solvents Corporation, Terre Haute, Ind., a corporation of Maryland I Application January'26, 1950,'SerialNo. 140,142

The present invention relates to anew crystalline form of riboflavin and to the method of preparing the same. More particularly, it re- H 7-Claims. ((31. 2609-2113) I I also discovered that riboflavin crystals of three definite and distinct forms may be prepared, the

' crystalline form obtained being dependent upon lates to a new crystalline form of riboflavin which 7 is .relatively soluble in water.

' Riboflavin, or vitamin B2, a vitamin of the B complex, is a naturally occurring vitamin and its deficiency isrecogni'zed as being the most widespread of any of the vitamins. Clinical 1 symptoms of riboflavin deficiency include anorexia, lassitude, keratitis, cheilosis, mydriasis and photophobia. As heretofore known and prepared, riboflavin is'only very slightly soluble in water and for that reason its use for hypodermic narily used in pharmaceutical or food products.

Riboflavin is variously reported in the literature to be soluble-in water to the extent of from 110 mg. per liter (U. S. Patent No. 2,440,050) to 150- 170 mg. per liter (U. S. Patent No. 2,407,624).

The art has therefore long sought for-a method of preparing more concentrated solutions of riboflavin which at the same time possess the very important properties of physiological activity and non-toxicity. Various means of producing riboflavin solutions have been tried, but they all possess disadvantages. Solubilizing agents have been used with some success as has been the methodof preparing soluble derivatives of riboflavin; Inaddition, numerous solvents have been proposed in which riboflavin is more soluble than inwater. For example, boron compounds, nicotinamide, gallic acid salts, urea, benzoic acid salts, L-tyrosine amide, veratryl alcohol, propylene glycol and vanillin have all been the subjects of numerous patents claiming these compounds as solvents for riboflavin, solubilizing agents or to form soluble reaction complexes with riboflavin. 1

None of the investigators has, however, succeeded-in'solving the problem presented by the insolubility of pure riboflavin in water alone. It has not been heretofore possible, therefore, to prepare aqueous solutions of pure riboflavin in concentrations .suflicient for hypodermic administration of good therapeutic dosesof riboflavin without the necessity of injecting very large volumes of liquid or injection of riboflavin in admixture with or solutions of partially toxic ma-it erials.

I It has been known that riboflavin may be made to crystallize to givea pure product. During the research work carried out on this investigation,

the method and conditions employed for crystallization. For convenience, the three crystalline forms of riboflavin will hereafter in this application be referred to as type A, type B, and type C. Type A is the known crystalline form and is only very slightly soluble in water. Type B has a solubility in water of about 200 mg. per liter provided about twice this amount is added to the water. The present invention is based upon the discovery that riboflavin can be prepared in a new crystalline form, type C, relatively soluble in water at all temperatures, and that this new form of riboflavin can be prepared in a simple, inexpensive and commercially feasible manner. This new form of riboflavin dissolves completely in water to the extent of 1200 mg. per liter at room temperatures.

The new crystalline form of riboflavin comprising my invention differs radically in physical appearance from the other two forms of riboflavin. Whereas type A consists of long, silky hair-like needles and type B consists of short, thin needles in bunches or shelves, my new crystalline form of riboflavin exists as clusters of short wide needles or plates.

Next to physiological activity, the solubility of riboflavin in water is the most important single property and the high solubility of my new type Criboflavin offers a definite means of identiflcation. Figurel shows in graphical'formthe solubilityqcharacteristics of the three crystal types of riboflavin. In this graph, there is plotted the mg. per liter of riboflavin dissolved (ordinate) against the quantities of the sample added (abscissa). All solubility measurements were carriedout by shakinga weighed amount oi the sample with'500 cc. of water-containing 0.5% acetic acid for 15 minutes atroom temperature. Referring to the graph of Figure 1. type A riboflavin shows acharacteristic solubility curve for a one component, solid phase. The maximum solubility isshown to be about mg. per liter. No further quantity of this form oi riboflavin dissolves when increasing amounts are added, thewexcess simply remaining as undissolved material.

Type B crystals give, to same extent at least, acharacteristic solubility curve of a two compo-v nentsolid phase mixture. However, microscopic examination and X-raydiffraction studies show that type B consists of only one type of crystal. The curve plotted from, the isolubility data obbe obtained with type B;

titles are addedadditional amounts go into solution but to a decreasing extent until a saturated solution is reached, after which the curve levels oiT. The ma gim um complete solubility of type B is shown'ftol bejBOmg; per literi but a solution of more than 200mg. per liter can, be obtainedby method developed by Paul L. Day and William J. Darby, and described in the article entitled "The Bioassay of Riboflavin appearing in Biological Symposia, volume XII, The Jacques Cattell Press, 1947. According to the method therein described 5.0 micrograms of the sample to be assayed is fed to rats daily for varying numbers of days. The wei'ghtsoflth'erats are recorded beforeeand after the tests. Identical tests are made "using a adding as much as 400 mg. per liter, leavinglo standard sample of riboflavin of known physioabout 200 mg. perliter undissolved. logical activity. To evaluate the activity of the As shown by the graph of F'igurel, typeTCJribQ- ,tested sample, the weight increases of the rats flavin crystals are much more highly solublethan fed the test sample are compared with the Weight either type A or B. Likewise,,type"A, thezsoluf increases of the'rats'fed the standard sample. bility'curveshows that type C is a purechem ical, 151The' results of the test with type C riboflavin, individual, but whereas type A is' completely sol- 'tabulatedinTable 11 below, show that the biouble only to the'extent of 60 mg.- per liter this logical activity ofthis new crystalline form of new type of riboflavin will dissolve completelyto 2 riboflavin is equal to or slightly higher than that the extent of 1200 mg. per liter; that is, it'is 20 of the U. S. P. Reference Standard used.

a TABLEII.

W v B i n 4 4 BodyWeightDuringf V 1 1 Depletion Period De lemon Daily Dose 1 ASSEY PBXiOdi(GmS)i m firm gigg 85??? 5 j "A Weight Initial l inal" Glidnge 1(SUPPLEMENT: U. s; PXRIBOFLAVIIN REFER NC sTA DA-m; i AZ (we; ams. days micrograms days 7' i T- -2s 45 29 r 5 2s. 79 152;.:-

;. ""2 44 .30 4 5 2s 73 144...; 3.23 45 32 5 '28 70 "124 s '23 44 32 5 :28. so 128 i: .a;

2a 44 s3 5 2s 83 129 46 3 23 45 33 5. 22s .378 I130 H.252 -23 44 v1 34 5 .22 104 188. ,84 23 4s "37 5 '28 114 172 58 23 44 51 L28 106 4149. .43;

- s9 4 147:4 ti-o (SUPPLEMENT: TYPE C'RIVBOFLAVIN) 2'3 29' "5 7 "28 83 "13s -55 "23 44 so 5 '28 73 =15? s4- 23 45 32 5 2s 150 -60 23 44 32 5 2s 88' "142 --54 23 I 45 32 5 .128: 198 150 1 52;; 23,, .44 33 5 28, no ;178 L68 .23 45 p 33 5 28' "7e 145'- s9 23 44 3s 5 28 .189 -139 .50 v23 45 36 5 2s. 97 166- .69, 3 43, 0 5 23 i '169 4; Axz'era'gei'. 9019 1533 f 62.5

timesas soluble as .ty-peA. compared-withitype B it is l5 times as soluble for complete solubility and a saturated 5511151011 5 6 times as concentratedyas themaximum concentration that can My'new type C crystallinefor m of riboflavin is also. much more soluble in. alcohols than .are

types-A and B. .IntableI,below, are-shownlthe results of solubilityitests at 25 o; withthe thre'ei 60 different" crystalline forms of riboflavin.

TABLELI ioiyaanaestrm j i j istwent 1 g 1 Since" the physiolo ical activity ofr iboflavifi is thie'ally importaflt property,- type "C riboflavin has-beens'ubmitted to rat fe'eding' te'sts The pro cedure e'mployed for these tests-was the accepted the filtrate should'have no morecolorthan 3 cc.

The U. S. Pharmacopoeia speciflcation'forriboflavin has required'that'the sample give a -negative test for lumiflavin. The" test prescribed is that when 25mg. of riboflavinisshaken'for5 minutes with 10 CC.'Of chloroform andfiltered,

of 0,1 N KzcrzOrdilutedto 1 liter-i -The-basis i01 this tes't is that-lumiflavin 'is' soluble"in-chloro form," thus 5 imparting a 001w when dissolved, Whereasriboflavin is insoluble in chloroform; Type C riboflavin gives a positive test forlumiflavin when tested in chloroform-as ordinarily sold on the market. Such chloroform contains about 1% alcohoLadded'as a preservative. The reason for the apparentpositive testis that type C riboflavin is so much more soluble than=;the other 4 forms of riboflavin :th'at suftleienti-s'of zthis riboflavin itself dissolvesin the chloroformzmona. taining alcohol) to impart"a.colorto th sOIutiom Thus, when the alcohol: is -removed:by washing the chloroform with waterandidrying outrthe water, a negative lumiflavin test is obtained;

alcohol-free chloroform, therefore, type 2C: riboflavin shows no signs of the presence,;of:=lumifla'.vin... 4 1 I Asa further confirmation of the conclusion that the apparent positive lumiflavin test obtained when using ordinary commercial chloroform, containing alcohol, is due to the actual solution of a small amount of the highly soluble riboflavin itself, the following experiment was carried out: An authentic sample. of type C riboflavin was shaken with chloroform to remove any-possible traces of lumiflavin. The crystals were filtered and dried. This sample gave a negative test for lumiflavin with alcohol-free chloroform, but still gave a positive test with the ordinary chloroform-containing alcohol.

As is known, X-ray .diflraction methods are capable of positively identifying crystalline phases. The difiraction angle, between. the X- rays incident upon the specimen, andthe set of planes of .interplanar spacing, d, is .givenby the well known Bragg relation A and d are generally expressed in Angstrom units (15:10" cm.). Thus, if is measured and i. is known, d can be readily computed.

For polycrystalline materials such as riboflavin, the spacings can be obtained by exposing a small amount of sample in a narrow beam of monochromatic X-rays of known wave length. The X-ray reflections from the planes of various spacings, d, strike a photographic film placed as a concentric cylinder around the sample. The various angles, 0, and the corresponding ds, are computed from the photographic record using Bragg relation. The set of spacing values, at, is unique for each crystalline phase, and these data, together with a measurement of the relative" intensities of the X-ray reflections from each set of planes providesa characteristic fingerprint or identification oi the phase.

In Figures 2, 3 and 4, and T bles III, and V,

the X-ray diffraction data obtained with samples of type A, type B and type C riboflavin are shown in graphical and tabular form. In the, graphs,

the relative intensity visually. estimated is plotted asordinate, and the sine otthe Bragg. angle,.0, or angle of. diffraction, is plotted :asthe abscissa.

As an alternatev iabcissa scale, the :interplanar spacings, d,.are aso marked below the 0 scale- For givensample; the'value of thesine 0 comtheseriesof reflections refer to the particular radiation used; Iron 1 any p'uted from the.photogram for radiation .=l.9373 A.) was used in this work. The basic values, :1, computed from the Bragg equation, dependently tabular form.

In Figures 2, 3 and 4, the intensity of a reflection is plotted as a vertical line of given height if the breadth of the reflection as seen on the film corresponds to the minimum line breadth characteristic of the camera. In some instances, however, the reflection is broader than the maximum width, and in these cases, a peak of finite width is plotted instead of a vertical'line. In" the photograms, line broadening 1 may resultfr'omf are representative of the sample ;in-' of the X-radiation employed, and'are generally preferred when the data' are given in I In Tables III. IV and V, the interplanar spacings (,d values) are given with the corresponding 3 spacing intensitites.

.TABL III Type A riboflavin crystals Spacing Spacing v 3 Intensity (Lug Intensity stroms) stroms) r 12.1 Veryfaint. 2.48 Veryiaint 10.2 Medium iainth 2.46."-.. Very, very faint: 9.2. Very, very faint. 2- D0. 8.5 Medium 2- Verylaint,difluse. 7.6 Very strong. 2.28. Faint clifiuse. 7.1..----. Faint. 2.22. Very, very faint. 67 Very, very faint. 2.15. Faint, difluse. 12 Very strong. 2.08. Very, very faint v Very, very faint. 2.05 Verytaint H 03 Medium''' 2.00 Very laintfidifluse. 86 Very, very faint, 1.96 Very faint.

d use. 1.9l7---.. Band. 72. Veryjaintt 1.89 Very, verytaint. .53. Medium strong. 1.86 Very, very iaint, .27. Faint, difluse. difluse. 02. Medium' 1.83 Do. .82. Very faint. I 1.723.---. Very faint, very (53g. lg leditum faint. 1 678 Vdifluse.

am e ver laint.+ 46. Medium. L643"... 150. y .30. Medium faint. y. y taint .22. Medium faint. use. .14. Very, very faint. 1. Very, very faint. .05. Very, very faint'*. Do. 2.96. Very strong. Do. 2.81. Faint diflnse. Very, very taint. 2.72. Faint. difiuSe. 2.64. Very taint, difluse. Do. 2.53 Very, very faint,

difiuse.

TABLE IV Type B riboflavin crystals spacing Spacing Intensity (Afar Intensity stroms) r lil Medium t aint 2.88 Faint Very, very faint. Medium faint. Medium strong. 2.75. Very, very faint. Strong". 2-67; Faint. D 2.57. Very aint+. Very, very Iaint D0.

Faint 2.38 Very faint.

. Do, I; 2.3 DO. Medium taint. 2.2 Very faint V 2.21 Very, very faint. Very, very faint. 2136- Veryfaint Very strong. 2.00. Faint. Very iaint 'difluse; 1.80. Very faint. Va -y m me 1.635 Very taint:v .7 Faint' 1.251 Very, very taint Medium strong. 1.153- Very, very faint. Mediumiaint Y TABLE V Type C riboflavin crystals Spacing Spacing l i Intensity. A Intensity 7 stroms) stroms) Very strong. 2- Very'ia'in'tfi diffuse. :Do. Very faint. Very, very faint. Very, 'verytaint."" Very, very t amt, dlf- Very, very iaint (use. Very, very faint. Medium faint, dif- Very, very !aint, dii'- I use.

Medium strong. -4 Very faint, difiuse. 5.72. Fa n 2.24 Very, very fainfl, 5.38. Medium faint. very diffuse. 5.02. Faint, difiuse'. Very,'very(aint.+ 4.46. Faintf, difiuse. 2- Very, very faint. 4.32. Very, very faint+. l. Do. 3.93. Very iaint diffuse. 1. ';Very, verylaintt.;- 3.,79. Medium taint. 1 1- ermyery faint. 3.58; Very, very iainth Do. 3.38-. strong ,1 j '1. Very, very faint, dif- 3. 2 6= Medium. fuse. '1 3.13. Very, very faint. 1.038(7)..." Very faint, difiuse;

Very, very faint, dit- Very, very I aintt; iuse.

'of lines with res'pectito intensity ratios.

' about 12.2and 1'0'.'8A., respectively.

mixtures ,lof these phases, and consequent overlapping :of .many lines, it is necessary to consider possible enhancement of the intensities,.and in practice the intensityratios of several pairs of lines should begcompared.

. v Thus; the mostcharacteristic X-ray difi'raction:

prQpertiesiofjtype Aarestrong "reflections with i For further identification, samples of the :various forms of riboflavin werestudied under the polarizing microscope; Provided the 'size of. the: crystals is sufficiently largeto enable the-.microscopist to make the necessary observations,:it is generallypossible to distinguish and identify phases from their optical activity. For riboflavin of types A andC sufficient data were obtained to .l distinguish these types 'in a -routine .manner.-J

Withiw .iType B, however crystallizesionly in the form of exceedingly smallcrystalsirom whichopti cal data are unobtainable. The size and appearancerof' type '3 crystals: provides a partial identification, byelimination.-

Ink Table VI, below, areLg-ive'n' the results oi? polarizing microscope I examinations of riboflavin-4 types Aand'C.

TABLE VI Type A Type C Refractive Index {delight-with elec trio veetonvibrating parallel to long axis. 7 ,Reiractive Index for light vibrating; A perpendicular-to-long axis; Sign oi Elongation Extinctionne".

interplanar spacings oiabout 7.6, 6.1 and 2.96 11.,

all nearly equal in intensity. Type Bis characterized by a strong broad peak or a double peak about 8.0 to 8.2 ALand strong-peaks at 6.2 and 3.83 a qua druplet of medium-strength reflections nearly equal in intensity and almost equally spaced at 5.0, 4.8, 4.5 and 4.3 A; respectively, and

a reflection of very high 'interplanar spacing about I 19 A. with moderate intensity.

Type Cis characterized byia pair of strong re-* flections nearly equal inintensity atabout 12.2} Iand,l0.8,:A.,respectively; a group-of unresolved reflections of :medium' intensities centered about.

6.1 A, andaa'. pair of'refictions-at338and see-A, "the1.3.3 8Aireflection .being about-twice as strong;

sme szze a;

I Ina mixture :of types A and Bj'th'e' strong ipeaks at 8 and 7.61 respectively, would both" 1 appear. but would not-befzcompletely resolved. The intensitiesotthe 6.l :and..6.2 A.. ee1 s would enhance each other. The relative intensity of the quadruplet of type B would be disturbed. The reflections at 10.2 and-2.96 A. would be usedto detect typeA. The reflections-Vat l9 and 3.83 A.

would be-Pusedto detect type B.

Ina mixture-'lof types A and .C, rthe reflections' at ilm-and". 96 2 woul d'be' used todetect type A;whilethe reflections:of the pair 'at 12.2 and 7 10.311. would-beusedto detect type C'.

Iii a miiitu'reo'f types B and c, th'ejre'fiec'ti'ons at l9,i i i .andf:3l83 A. would be used to-"detect type: is, and"th'ej;pair "at 12:25am atfl0i8i would be; used todetect WDe CL- v .It' can therefore beseen' that in asan'iplewhich;

V is 'known" to ;}contain riboflavin, the 1 presence. of;

the; relatively soluble type Criboflavin can be".

easily determin'ed by the characteristic pair or" strong. reflections nearly equal in intensity at;' gramg p l fla riboflavzlh.- Ezicell'ent results when aqueous solutions otammonium hydroxide;

Long Needles With Very (lrossjse'ction No Twinning? grams of riboflavin Positive... Negative. 1 Parallel 1 Y Variesirom-about33 to (fies meas ur'ed'from the long edge of crystal;

- vibrating nearly 'parallek to long axis ofjthe crystal and orange for light vibrating nearly perpendic ,ular to thelong axis of the crystal) 1 The nevv crystalline form of riboflavin which -comprises my invention can be prepared by dis solving crude riboflavin 'iniaqueous alkaline solu= tion to give a riboflavin concentration. of between 10 and 30 grams-per liter. The solution is filtered andacidifiedwith' an 'acid'jand' riboflavin -i-m-' mediately crystallizes in the form of 'bunches r rosettes of well defined; plate-like needles. The

crystals are thenfiltered, washed-and*dried---in -a vacuum oven. 7

The purity of the crude riboflavin isnotthe factor which: determines i the 1 crystallineformobtained 'inthe crystallization-step; I have-found that: riboflavin of any. purity? can be used; pro-1;

vided. suflicint quantities" are added to give .an'i alkaline; solution 'containing -;-l:ietween '10 :and: 30 per-'liteig: at room temperaetures; However; a reasonably pure crude; prod-:- uct should be employed 'as the "starting material so as; to prevent crystallization of a final prode uct containing large amounts'of impuritiesiflI- have found that crude riboflavin of from V to purity gives afinal product ofyeryhiglalv purity.

Any aqueous. alkaline solution w hich willfdis solve fromlO to 30 grams per liter of riboflavinv at room ltemperatures ..can be used. The alkali metal hydroxidesare particularly. adaptable for sincevery dilute .solutionsof 't'he ch ydroxides; readily dissolve vsuilicient qua-ntitiesnof .arealso obtained are..useitoldissolve the riboflavin... -Iii addi'tidm aqueous solutions;- of. any. amine wh'ich'will hissolve sufficient fibbflaviri to. give .3. concentration. v of v .1( l'-l30 g rarnsj=per liter can be used.;. The aliphatic Qaminesiarel particularly. useful.iorihispurmse. V -1131; I 'rrhe' aqueous alkaline solution musrbeiorisuffi cient'; 'strength'. to. dissolve. between. about 10931) "I The iiformalitylof I per liter;

. 5 the "alkaline solution sh'ou d'fb kept as law as" critical.

possibleg'but still dissolve sufficient quantities of "riboflavin.

While the concentration of" riboflavin in the aqueous-alkaline solution must be between and 30 grams per-liter to produce typeC'crystals at f room temperatures, (20 to 25 C.),' considerably lesser concentrations may be employed :if the solution is cooled to about 10 C. before the acidi- -fflcation'.- Thus, if the solution is cooled before I acidifying"; the concentration of riboflavin in the aqueous alkaline-solution can be as=1ow as 2 to 3 -the solution so as to'separate any non-soluble impurities present in the. startingycr'ude' riboflavin.

1 After filtration; acid isadded to the aqueous alkaline .solution containing riboflavin in quantities'sufiicient'to reduce the 'pHbelow about"9.0,

and preferably below about 6.0. Any acid may be usedfor this step,particularly good results being obtained with sulfuric, nitric, hydrochloric, phosphoric, and'acetic acids. When the pH of the solution reaches below about 9.0 crystals of ,ournew type C riboflavin immediately separate. The crystals are then filtered, washed and dried.

It is necessary that the crystals be washed to remove excessive amounts of salts which are found in the wet cake following filtration. My new type crystal is a'highly metastable form of riboflavin and has a tendency to revert to. a crystalline form of lower solubility during the washing step. This reversion tendency is not apparent during the crystallization and filtration steps.

I have found that the crystals can be washed with tertiary butyl alcohol without any reversion of the crystalline form taking place. Other organic solvents which we have tried for the washing treatment have all caused considerable ouantities of the crystals to revert to a form of lower solubility. Water can also be used to wash the crystals, but without the high degree of success experienced with tertiary butyl alcohol. The crystals can be consistently washed with water, without reversion taking place, provided the temperature is maintained below about 10 C. With tertiary butyl alcohol, however, refrigeration is not necessary. Washing the filter cake with tertiary butyl alcohol at room temperatures has ,in every instance given a low ash product completely free from any signs of a lower soluble form of riboflavin.

For a more complete understanding of the method of preparation of my new crysta ine form of riboflavin, reference will now be made to certain specific examples. It will be understood, however, that this invention is not to be restrict- .ed in any wav by these specific examples since they are merely illustrative and are not definitive of the broad aspects of the present invention.

EXAMPLE I I crystals were then removed on a suction funnel, 1 washed with a little cold water and 'dried in a ash and dissolved completelyin One hundred and fifty i grams ofcrudefribofilter cake washedwith-waten The-filtrate and rated by filtration on a, suction funnel -washed' with tertiary butyl alcoholgunti the wash combined were dilutedto 6000 ml .,-thusgiving a'solution of;23.8 grams, of riboflavin per liter in 0.08 N-sodium hydroxide solution. Thissolu tion was acidified slowly'w ith 2 N sulfuriqacid, crystallization starting as-the pI-I-reached about 8.0. The acidification was continuedtoa pl l: of 4.0. After fifteen minutes the crystals -weresepaand washings were free from the sulfate iom l The filter cake was; dried in ava-cuumoven at 70 C.

I pletely ;at a level of 1200 mg perliterg The resulting;product-assayed: 100%-riboflavin, was essentially; free from ash and dissolved. oom- 7 EXAMPLE III" I Inthis example the procedure outlined "i V I ample II was followedlexactly,"except the the riboflavin crystals were washed withlwatrlat a temperature of about 10 .C., In this example the resulting product assayed 100%' ribo'flavin andjdi'ssolved completely at. a level 9:; 1'20 per liter. While some particular embodiments of this invention are shown above, it wil1 be understood, of course, that the invention is not to be limited thereto, since any modification may be made, and it is contemplated, therefore, by the appended claims, to cover any such modifications as fall within the true spirited scope of this invention.

Iclaim:

1. A new crystalline form of riboflavin, as described, characterized by the following properties: (a) refractive index parallel to the elongation of crystals of less'than about 1.538 and perpendicular to elongation of above 1.74, (b) negative sign of elongation, (c) extinctionvarying from about 33 to 0 as measured from long edge of crystals, (d) strong pleochroism, (e) common twinning of crystals, (fl solubility .in water of about 1200 mg. per liter at 25 C., (g) X-ray diffraction properties characterized by a pair of strong reflections nearly equal in intensity at about 12.2 and 10.8 A, respectively, a group of unresolved reflections of medium intensities centered about 6.1 A. and a pair of refiections at 3.38 and 3.26 A, the 3.38 A. reflection being about twice as strong as the'3.26 A.

2. The process for preparing a new crystalline form of riboflavin which comprises, dissolving from 10-30 grams of riboflavin per liter in an aqueous alkaline solution, adding acid to said solution, separating the crystals of riboflavin which form, washing said crystals with a member selected from the group consisting of water at a temperature below about 10 C. and tertiary butyl alcohol, and drying said riboflavin crystals.

3. The process for preparing a new crystalline form of riboflavin which comprises dissolving riboflavin in an aqueous alkaline solution selected from the group consisting of alkali metal and 

1. A NEW CRYSTALLINE FORM OF RIBOFLAVIN, AS DESCRIBED, CHARACTERIZED BY THE FOLLOWING PROPERTIES: (A) REFRACTIVE INDEX PARALLEL TO THE ELONGATION OF CRYSTALS OF LESS THAN ABOUT 1.538 AND PERPENDICULAR TO ELONGATION OF ABOVE 1.74, (B) NEGATIVE SIGN OF ELONGATION, (C) EXTINCTIONVARYING FROM ABOUT 33* TO 0* AS MEASURED FROM LONG EDGE OF CRYSTALS, (D) STRONG PLEOCHROISM, (E) COMMON TWINNING OF CRYSTALS, (F) SOLUBILITY IN WATER OF ABOUT 1200 MG. PER LITER AT 25* C., (G) X-RAY DIFFRACTION PROPERTIES CHARACTERIZED BY A PAIR OF STRONG REFLECTIONS NEARLY EQUAL IN INTENSITY AT ABOUT 12.2 AND 10.8 A., RESPECTIVELY, A GROUP OF UNRESOLVED REFLECTIONS OF MEDIUM INTENSITIES CENTERED ABOUT 6.1 A. AND A PAIR OF REFLECTIONS AT 3.38 AND 3.26 A., THE 3.38 A. REFLECTION BEING ABOUT TWICE AS STRONG AS THE 3.26 A. 